27 research outputs found
The role of Mcl-1 in S. aureus-induced cytoprotection of infected macrophages
As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NF B activation and subsequent IL-6 secretion, since the inhibition of both NF B and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases
PD-1 checkpoint inhibition enhances the antilymphoma activity of CD19-CAR-iNKT cells that retain their ability to prevent alloreactivity.
Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties.
iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings.
In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses.
In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT
Retromer and Its Role in Regulating Signaling at Endosomes.
The retromer complex is a key element of the endosomal protein sorting machinery being involved in trafficking of proteins from endosomes to the Golgi and also endosomes to the cell surface. There is now accumulating evidence that retromer also has a prominent role in regulating the activity of many diverse signaling proteins that traffic through endosomes and this activity has profound implications for the functioning of many different cell and tissue types from neuronal cells to cells of the immune system to specialized polarized epithelial cells of the retina. In this review, the protein composition of the retromer complex will be described along with many of the accessory factors that facilitate retromer-mediated endosomal protein sorting to detail how retromer activity contributes to the regulation of several distinct signaling pathways
A genome-wide RNAi screen identifies MASK as a positive regulator of cytokine receptor stability
Cytokine receptors often act via the Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway to form a signalling cascade that is essential for processes such as haematopoiesis, immune responses and tissue homeostasis. In order to transduce ligand activation, cytokine receptors must dimerise. However, mechanisms regulating their dimerisation are poorly understood. In order to better understand the processes regulating cytokine receptor levels, and their activity and dimerisation, we analysed the highly conserved JAK/STAT pathway in Drosophila, which acts via a single receptor, known as Domeless. We performed a genome-wide RNAi screen in Drosophila cells, identifying MASK as a positive regulator of Domeless dimerisation and protein levels. We show that MASK is able to regulate receptor levels and JAK/STAT signalling both in vitro and in vivo. We also show that its human homologue, ANKHD1, is also able to regulate JAK/STAT signalling and the levels of a subset of pathway receptors in human cells. Taken together, our results identify MASK as a novel regulator of cytokine receptor levels, and suggest functional conservation, which may have implications for human health
Integration of JAK/STAT receptor-ligand trafficking, signalling and gene expression in Drosophila melanogaster cells
The JAK/STAT pathway is an essential signalling cascade required for multiple processes during development and for adult homeostasis. A key question in understanding this pathway is how it is regulated in different cell contexts. Here we have examined how endocytic processing contributes to signalling by the single cytokine receptor, Domeless, in Drosophila melanogaster cells. We identify an evolutionarily conserved di-Leu motif that is required for Domeless internalisation and show that endocytosis is required for activation of a subset of Domeless targets. Our data indicate that endocytosis both qualitatively and quantitatively regulates Domeless signalling. STAT92E, the single STAT transcription factor in Drosophila, appears to be the target of endocytic regulation and our studies show that phosphorylation of STAT92E on Tyr704, while necessary, is not always sufficient for target transcription. Finally, we identify a conserved residue, Thr702, which is essential for Tyr704 phosphorylation. Taken together, our findings identify previously unknown aspects of JAK/STAT pathway regulation likely to play key roles in the spatial and temporal regulation of signalling in vivo
Role of endosomal sorting in IFN I receptor trafficking and JAK/STAT signaling.
La voie JAK / STAT est une voie majeure de signalisation intracellulaire, activĂ©e par plusieurs cytokines, y compris par les interfĂ©rons (IFNs). Mon laboratoire a prĂ©cĂ©demment Ă©tabli que l'endocytose et le trafic du rĂ©cepteur de lâIFN alpha/beta (IFNAR) jusquâĂ l'endosome prĂ©coce, joue un rĂŽle clĂ© dans lâactivation de la voie de signalisation JAK/STAT et dans les activitĂ©s antivirales et antiprolifĂ©ratives induites par les IFNs de type I. Le rĂ©cepteur de lâIFN de type I se compose de deux sous-unitĂ©s: IFNAR1 et IFNAR2. Durant le trafic du rĂ©cepteur vers l'endosome prĂ©coce, les deux sous-unitĂ©s prennent des itinĂ©raires diffĂ©rents - IFNAR1 est ubiquitinĂ© et dĂ©gradĂ© au lysosome tandis que IFNAR2 est recyclĂ© vers la membrane plasmique. Les mĂ©canismes molĂ©culaires permettant un trafic diffĂ©rent des IFNAR1 et IFNAR2 restent mal compris.Jâai tout dâabord Ă©tudiĂ© le trafic intracellulaire des sous-unitĂ©s IFNAR1 et IFNAR2 et jâai pu montrer que les protĂ©ines Rab4 et Rab11 sont nĂ©cessaires au recyclage dâIFNAR2 Ă la membrane plasmique. Par la suite, jâai identifiĂ© le complexe rĂ©tromĂšre endosomal - VPS26A/VPS29/VPS35 comme un partenaire dâinteraction avec IFNAR2 nĂ©cessaire pour son recyclage. En outre, jâai montrĂ© que le complexe rĂ©tromĂšre contrĂŽle la sĂ©paration spatiotemporelle des sous-unitĂ©s IFNAR1 et IFNAR2 au niveau de l'endosome prĂ©coce. En effet, la dĂ©plĂ©tion du complexe rĂ©tromĂšre entraine une prolongation de lâassociation endosomale de deux sous-unitĂ©s et une prolongation de la signalisation JAK/STAT induite par lâIFN tant pour la rĂ©ponse prĂ©coce (phosphorylation de STAT1) que la rĂ©ponse Ă plus long terme (expression des gĂšnes stimulĂ©s par l'IFN). Des rĂ©sultats en cours de publication par lâĂ©quipe montrent un rĂŽle dâESCRT-0 dans lâinitiation de la signalisation JAK/STAT. Jâai montrĂ© que le rĂŽle de la machinerie ESCRT dans ce processus est limitĂ© au complexe ESCRT-0. Les sous-complexes en aval dâESCRT-0, bien que nĂ©cessaires Ă la dĂ©gradation dâIFNAR1, ne sont pas impliquĂ©s dans lâactivation de la voie JAK/STAT.En conclusion, ces rĂ©sultats nous ont permis dâĂ©tablir un modĂšle dans lequel le retromĂšre joue un rĂŽle clef dans la rĂ©gulation spatio-temporelle du tri endosomal dâIFNAR et de la signalisation JAK/STAT au niveau de lâendosome prĂ©coce. AprĂšs la sĂ©paration rĂ©tromĂšre-dĂ©pendante des sous-unitĂ©s du rĂ©cepteur et la terminaison de la signalisation JAK/STAT, la dĂ©gradation lysosomale dâIFNAR1 est assurĂ©e par la machinerie ESCRT en aval dâESCRT-0.The JAK/STAT pathway is a major intracellular signaling pathway that is activated by several cytokines including interferons (IFNs). My laboratory has previously established that endocytosis and trafficking of the IFN alpha/beta receptor (IFNAR) to the early endosome is key for the activation of JAK/STAT signaling and for the antiviral and antiproliferative activities induced by type I IFNs. The functional type I IFN receptor - IFNAR - consists of two subunits: IFNAR1 and IFNAR2. Upon endocytosis to the early endosome, both subunits take different trafficking routes â IFNAR1 is ubiquitinated and degraded in the lysosome, whereas IFNAR2 recycles back to the plasma membrane. The molecular mechanisms behind the separation of IFNAR1 and IFNAR2, as well as their distinct trafficking routes remain poorly understood.First, I studied the intracellular trafficking of IFNAR1 and IFNAR2 subunits and showed the requirement for Rab4 and Rab11 in IFNAR2 recycling to the plasma membrane. Next, I identified the endosomal retromer complex â VPS26A/VPS29/VPS35 as an IFNAR2 interacting partner, required for IFNAR2 recycling. Additionally, I was able to show that retromer controls the spatiotemporal separation of IFNAR1 and IFNAR2 subunits at the early endosome. Indeed, retromer depletion resulted in prolonged endosomal association of both subunits and prolonged activation of IFN-induced JAK/STAT signaling, at both early (STAT1 phosphorylation) and longer time response (IFN-stimulated gene expression). Unpublished results of our team indicate the role of ESCRT-0 in initiation of the IFN-mediated JAK/STAT signaling. I found that role of the ESCRT machinery in this process is limited to the ESCRT-0. ESCRT subcomplexes downstream of ESCRT-0, although required for IFNAR1 degradation, are not involved in activation of the JAK/STAT pathway.In conclusion, these results permit us to draw a model in which the retromer is a key spatiotemporal regulator of IFNAR endosomal sorting and the JAK/STAT signaling at level of the early endosome. Once retromer-mediated subunits separation is accomplished and JAK/STAT signaling is terminated, ESRCT machinery downstream to ESCRT-0 mediates IFNAR1 lysosomal degradation
Yugoslavian partisan comic on the example of the analysis of the album Partisans (vol. I) ÄorÄe Lebovicia and Julio Radilovicia
Historia jugosĆowiaĆskiej partyzantki wciÄ
ĆŒ cieszy siÄ sporym zainteresowaniem. SpoĆrĂłd wielu rĂłĆŒnych form jej popularyzacji na szczegĂłlnÄ
uwagÄ zasĆuguje komiks. W niniejszej pracy zaprezentowany zostanie jeden z nich -seria komiksĂłw zebrana do albumu "Partyzanci" ÄorÄe Lebovicia i Julio Radilovicia-Julesa, ktĂłra od poczÄ
tku przeznaczona byĆa dla odbiorcy z Europy Zachodniej, przedstawiaĆa partyzantĂłw w odmienny sposĂłb niĆŒ jugosĆowiaĆski komiks. Aby zrozumieÄ tÄ rĂłĆŒnicÄ w sposobie prezentacji ich historii, trzeba przywoĆaÄ istotny w tym wypadku kontekst polityczny i kulturowy oraz wskazaÄ na specyfikÄ jugosĆowiaĆskiego komiksu.The history of Yugoslavian partisans is still very popular. Among many different forms of its popularization, comics deserves special attention. In this work will be presented one of them - a series of comics collected in the album "Partisans" by ÄorÄe Lebovic and Julio Radilovic-Jules, which from the beginning was intended for a reader from Western Europe, presented partisans in a different way than the Yugoslavian comic. To understand this difference in the way their stories are presented, it is necessary to recall the relevant political and cultural context , and point to the specificity of the Yugoslavian comic book